Blood component therapy, made possible by the development of a closed system of multiple plastic collecting bags and high speed refrigerated centrifuges, is one of the most important advances in the history of blood transfusion practices. The rationale for use of specific fractions of blood is that blood contains a number of differently formed elements as well as various plasma proteins and constituents which have many functions. Thus, a single donation of a unit of whole blood can provide red blood cells, platelets, plasma, and cryoprecipitated factor VIII - fibrinogen concentrate. Pheresis procedures are able to supply large quantities of granulocytes, platelets, and plasma from single donors. Plasma can be chemically fractionated to provide albumin or plasma protein fraction, factor VIII concentrate, factor IX complex and immune serum globulin.
The rationale for using blood components is that a patient usually requires replacement of only a specified component (See: Greenwalt et al: General Principles of Blood Transfusion, A.M.A. Editorial Board, 1978). Remaining components can be then used to treat patients who require other specific components thereby allowing several patients to benefit from each unit of donated blood thereby maximizing the benefit realizable therefrom.
Factor IX complex is a lyophilized pooled plasma derivative rich in Factors IV, VII, IX and X. It is an alternative to plasma therapy. It supplies vitamin K-dependent clotting factors in a much smaller volume than plasma but with a significantly higher hepatitis risk.
Factor IX containing concentrates are a unique and highly valuable blood product which are life-saving when used to control bleeding in patients suffering with factor IX deficiency (Hemophilia B). These products have also been used to treat those patients inflicted with Hemophilia A having inhibitors although clinical verification of this application is still in progress. Factor IX containing concentrates are also used to arrest serious hemorrhages or to avert operative and post operative bleeding in patients with other congenital clotting factor deficiencies and for multiple factor deficiency induced by an overdose of warfarin-type drugs, i.e., oral anticoagulants.
Commercial concentrates of factor IX have been previously prepared using ion exchange resins to bind vitamin K-dependent clotting factors and separate these proteins from the bulk of other plasma proteins. These clotting factor concentrates are then eluted from the resin and vialed for therapeutic use without further purification. Such concentrates tend to be thrombogenic probably because they contain extraneous vitamin K-dependent clotting factors and/or phospholipid. Further, such concentrates have been a suspected vehicle in the transmission of viral diseases including hepatitis and acquired immune deficiency syndrome ("AIDS"). Further, crude concentrates of factor IX are not stable in vitro and therefore cannot be used for constant infusion therapy which limits its value in chronic replacement therapy.
Recent efforts to create factor IX using a recombinant DNA approach have been frustrated by the difficulty encountered in separating factor IX from culture supernatants with currently accepted techniques. (See: Anson DS, Austen DEG, and Brownlee GG. "Expression of active human clotting factor IX from recombinant DNA clones in mammalian cells." Nature 1985;315:683-685; de la Salle H, Altenburger W, Elkaim R, Dott K, et al. "Active .gamma.-carboxylated human factor IX expressed using recombinant DNA techniques." Nature 1985;316:268-270; and Busby S, Kumar A, Joseph M, Halfpap L, Insley M, et al. "Expression of active human factor IX in transfected cells." Nature 1985;316:271-273).
Thus a very special need exists for the development of means and methods for the manufacture and isolation of highly purified factor IX by affinity chromatography and which can thereafter be formulated into a potent, quick-acting, therapeutic blood product which is stable in vitro and which provides effective relief for patients encountering a critical bleeding incident.
It is to the resolution of that need that the present invention is directed which provides a cell line and method of producing a unique antibody therefrom which antibody is exceptionally suited for purifying factor IX complex and thereby permits the production of a high purity factor IX therapeutic blood product therefrom.